Enterococcus spp. is a common commensal microorganism, however, some strains can cause opportunistic infections in humans. Treatment of Enterococcus faecium-related endocarditis, urinary and genital tract infections, meningitis, septicemia, and even neonatal sepsis is often complicated by antibiotic resistance. The spread of multi-resistant bacterial strains has renewed interest in phage therapy, which has many advantages: Its advantages include a much lower frequency of resistance development compared to antibiotics and strict specificity, which allows affecting of only their target microbes without disturbing necessary microbiome. We isolated and characterized a virulent bacteriophage which is active against Enterococcus faecium clinical strains. The phage, which was designated as vB_GEC_EfS_9 was studied in terms of its growth pattern and adsorption rate, as well as its host range. The whole genome of the phage was sequenced and analyzed. Obtained results indicate that phage vB_GEC_EfS_9 is a virulent phage which has a very good potential for therapeutic use against strains of E. faecium.

Arboviruses are transmitted by arthropods (arthropod-borne virus) which can be mosquitoes or other hematophagous arthropods, in which their life cycle occurs before transmission to other hosts. Arboviruses such as Dengue, Zika, Saint Louis Encephalitis, West Nile, Yellow Fever, Japanese Encephalitis, Rocio and Murray Valley Encephalitis viruses are some of the arboviruses transmitted biologically among vertebrate hosts by blood-taking vectors, mainly Aedes and Culex sp., and are associated with neurological, viscerotropic, and hemorrhagic reemerging diseases, posing as significant health and socioeconomic concern, as they become more and more adaptive to new environments, to arthropods vectors and human hosts. One of the main families that include mosquito-borne viruses is Flaviviridae, and here, we review the case of the Flavivirus genus, which comprises the viruses cited above, using a variety of research approaches published in literature, including genomics, transcriptomics, proteomics, metabolomics, etc., to better understand their structures as well as virus-host interactions, which are essential for development of future antiviral therapies.

Orf virus (ORFV) is the type species of the Parapoxvirus genus that belongs to the Poxviridae family. Type I interferons (IFN) are critical in the host defence against viruses. They induce hundreds of interferon stimulated genes (ISGs) many of which have an antiviral role. The ability of ORFV to modulate type I IFN production was undertaken to investigate whether ORFV could inhibit IFN-β expression via dsRNA dependant signalling pathways. HEK293 cells are known to lack DNA pattern-recognition receptors and Toll-like receptors however, they do express the cytosolic dsRNA receptors RIG-I and MDA5. HEK293 cells were shown to produce high levels of IFN-β when cells were stimulated with poly(I:C) and this was shown to be predominantly via RIG-I-dependant signalling as confirmed by siRNA knock-down of RIG-I. Further we showed that HEK293 cells are permissive for ORFV and caused potent inhibition of IFN-β transcription when cells were stimulated with poly(I:C) post-viral infection. Studies using heat inactivated ORFV suggested that de novo synthesis of early genes was required. In addition our findings showed that the ORFV encoded factor ORF020, that is known to bind dsRNA, is involved in antagonising IFN expression. Overall, this study has shown for first time the ability of ORFV to counteract type I IFN expression by antagonising dsRNA-activated RIG-I signalling.

Japanese encephalitis virus (JEV) causes the most commonly diagnosed viral encephalitis in Asia. JEV is a highly neurotropic flavivirus that can replicate efficiently in the brain. Axl belongs to the TAM (Tyro3, Axl, Mer) family, a group of tyrosine kinase receptors involved in the viral entry, micked as apoptotic bodies and regulation of innate immunity. However, the underlying mechanisms on its regulation in the neurons for JEV are unclear. Here, we found that Axl was upregulated in neurons after JEV infection. Unexpectedly, Axl deficient (Axl-/-) mice were more susceptible to JEV infection with increased viral loads in neurons. The RNA-sequencing analysis between the wild type neurons and Axl-/- neurons infected with JEV showed that many interferon-stimulated genes were downregulated in the Axl-/- neurons which innate immunity was attenuated largely. The rescue experiment in Axl-/- neurons indicated that Axl may be positively involved in the regulation of antiviral immunity. Taken together, our data demonstrated that Axl may play an antiviral role in JEV replication within neurons by modulating neuronal innate immunity.

Classical swine fever virus (CSFV) infection causes a severe disease of pigs, resulting in significant economic losses. The CSFV NS4B protein is crucial for viral replication and pathogenicity. Interleukin 8 (IL-8), a main chemokine, is induced by multiple cell types and plays an essential role in host defense mechanisms against numerous viruses. It has been reported that NS4A of CSFV is involved in the induction of IL-8 expression in swine umbilical vein endothelial cells. However, the effect of CSFV NS4B on IL-8 expression is unknown. In this study, we showed that CSFV NS4B inhibited IL-8 expression in porcine alveolar macrophages (PAMs), and NS4B inhibited mitochondrial antiviral signaling protein (MAVS)-induced IL-8 expression. Moreover, CSFV NS4B interacted with MAVS. However, NS4B did not alter MAVS expression. Subsequently, we demonstrated that IRF3 knockdown or NF-κB inhibition reduced MAVS-induced IL-8 expression. Furthermore, the IRF3 and NF-κB pathways were activated by MAVS expression. However, CSFV NS4B inhibited MAVS-mediated NF-κB activation and IRF3 expression. Finally, CSFV NS4B inhibited IRF3 expression. Our findings reveal that CSFV NS4B interacts with MAVS and inhibits IL-8 expression by blocking the activation of IRF3 and NF-κB. Taken together, this study provides insights into the mechanism of NS4B-inhibited IL-8 expression.

A novel phage vB_VpP_DE17, which infects Vibrio parahaemolyticus, was isolated from the sewer of the Huangsha aquatic market in Guangzhou. Transmission electron microscopy indicated that DE17 had an icosahedral head (47 ± 2 nm diameter) and a short, non-contractile tail (17 ± 2 nm). The genome of DE17 was a double-stranded linear DNA with a length of 43,397 bp and GC content of 49.23%. In total, 49 putative open reading frames (ORFs) were predicted and could be divided into six modules: DNA metabolism, lysis, packaging, structure, additional function, and hypothetical proteins. Taxonomic analysis revealed that the phage belonging to the genus of Maculvirus, Autographivirinae subfamily, Podoviridae family. DE17 had a short latent period of 5 min with burst size of 80 pfu/cell. Its optimum temperature and pH ranges were 4 °C-50 °C and 5-10, respectively; it was completely inactivated after 20 min of ultraviolet irradiation. No transfer RNA (tRNA), virulence associated, or antibiotic resistance genes were identified. Bacterial challenge test revealed that DE17 had a certain inhibitory effect on V. parahaemolyticus within 6 h. Characterization, genomic analysis and in vitro antibacterial assays of DE17 will further enhance our understanding of phage biology and diversity.

Expanding on our previous work, this study used transcriptome analysis of RNA sequences to investigate the various factors that contributed to either inducing apoptosis that resulted in cell death or promoting the survival of African horse sickness virus serotype 4 (AHSV4)-infected horse peripheral blood mononuclear cells (PBMC) after 24 h. Apoptosis is a host defense mechanism that prevents virus replication, accumulation and spread of progeny viruses. AHSV4-infected PBMC were killed via the intrinsic and the perforin/granzyme pathways of apoptosis during the attenuated AHSV4 (attAHSV4) in vivo primary and secondary immune responses. Trained innate immunity played an important role in circumventing viral interference that resulted in the elimination of AHSV4-infected PBMC through the intrinsic and the extrinsic pathways of apoptosis during the virulent AHSV4 (virAHSV4) in vitro secondary immune response. Oxidative stress in conjunction with IRE1α pro-apoptotic signaling played a major role in the induction of the intrinsic pathway of apoptosis and cytotoxic lymphocytes induced the perforin/granzyme or extrinsic pathways of apoptosis. In contrast, AHSV4-infected PBMC survived during the virAHSV4 in vitro primary immune response, which allows unrestrained viral replication. The virAHSV4 interference with the innate immune response resulted in impaired NK cell responses and delayed immune responses, which together with the antioxidant defense system promoted AHSV4-infected PBMC survival.

The second wave of COVID-19 caused by severe acute respiratory syndrome virus (SARS-CoV-2) is rapidly spreading over the world. Mechanisms behind the flee from current antivirals are still unclear due to the continuous occurrence of SARS-CoV-2 genetic variants. Brazil is the world's second-most COVID-19 affected country. In the present study, we identified the genomic and proteomic variants of Brazilian SARS-CoV-2 isolates. We identified 16 different genotypic variants were found among the 27 isolates. The genotypes of three isolates such as Bra/1236/2021 (G15), Bra/MASP2C844R2/2020 (G11), and Bra/RJ-DCVN5/2020 (G9) have a unique mutant in NSP4 (S184N), 2'O-Mutase (R216N), membrane protein (A2V) and Envelope protein (V5A). A mutation in RdRp of SARS-CoV-2, particularly the change of Pro-to Leu-at 323 resulted in the stabilization of the structure in BRA/CD1739-P4/2020. NSP4, NSP5 protein mutants are more virulent in genotype 15 and 16. A fast protein folding rate changes the structural stability and leads to escape for current antivirals. Thus, our findings help researchers to develop the best potent antivirals based on the new mutant of Brazilian isolates.

The plant pathogen Exobasidium gracile, which belongs to the basidiomycetous genus Exobasidium, can lead to swollen and thicker leaves of C. oleifera. To our knowledge, there have been no reports of mycoviruses infecting Exobasidium gracile. This study characterized three mycoviruses coinfecting the plant pathogen Exobasidium gracile strain Z-1. Based on phylogenetic and genomic analyses, E. gracile strain Z-1 was infected two putative Totiviruses designated Exobasidium gracile Totivirus 1 (EgTV1) and Exobasidium gracile Totivirus 2 (EgTV2) and a putative Zybavirus of the family Amalgaviridae defined Exobasidium gracile Zybavirus 1 (EgZV1). Similar to the genomic organization of other Totiviruses, the EgTV1 and EgTV2 genomes are composed of one dsRNA segment that exhibits two large ORFs encoding a CP (capsid protein) and an RdRp (RNA-dependent RNA polymerase), respectively. Moreover, EgTV1 and EgTV2 genomes with a candidate -1 slippery heptamer sequence were discovered between CP and RdRp, respectively. Similar to other Zybaviruses of the family Amalgaviridae, the EgZV1 genome is composed of one dsRNA segment that contains two large ORFs encoding an unknown protein and an RdRp. In addition, the EgZV1 genome has a candidate +1 slippery heptamer sequence between an unknown protein and RdRp.

Pepper mild mottle virus (PMMoV) infects pepper plants and induces severe yield losses in China. However, the molecular interaction between PMMoV and pepper plants is largely unknown. RNA silencing is a eukaryotically conserved mechanism against viruses mediated by virus-derived small interfering RNAs (vsiRNAs) in plants. In this study, the profiles of vsiRNAs from PMMoV in infected pepper plants were obtained by high-throughput sequencing. The results showed that vsiRNAs were predominantly 21 and 22 nucleotides (nts) in length, and had a U bias at the 5'-terminal. The single-nucleotide resolution maps revealed that vsiRNAs were heterogeneously distributed throughout PMMoV genomic RNAs and hotspots of sense and antisense strands were mainly located in the RdRp and CP coding regions. The host transcripts targeted by vsiRNAs were predicted and they are mainly involved in physiological pathways related to stress response, cell regulation, and metabolism process. In addition, PMMoV infection induced significant up-regulation of CaAGO1a/1b/2, CaDCL2 and CaRDR1 gene transcripts in pepper plants, which are important components involved in antiviral RNA silencing pathway. Taken together, our results suggest the possible roles of vsiRNAs in PMMoV-pepper interactions.

In this study, we present the complete, annotated genome of a new member of the Tequatrovirus (T4-like) genus, Escherichia phage vB_EcoM_C2-3. This phage has an isometric head (92 nm in diameter) and a contractile tail (114 nm in length). Its genome consists of a linear, double-stranded DNA of 167,069bp with an average G+C content of 35.3%. There are 267 predicted genes, of which 125 encode functional proteins, including those for DNA replication, transcription and packaging, phage morphogenesis and cell lysis. Neither genes involved in the regulation of lysogeny nor antibiotic resistance genes were identified. Based on our results, its genomic features provide valuable insights into the use of a potential biocontrol agent, as Escherichia phage vB_EcoM_C2-3 exhibited lytic activity against E. coli, including multidrug-resistant strains.

Erratum in Virus Res. 2023 Jan 15;324:199013.

Antibody profile of pigs naturally infected with a virulent African swine fever virus (ASFV) strain under field conditions was studied. Twenty-three serum samples were collected from pigs surviving a natural ASFV infection: 17 samples from finishing pigs (∼7 months old) and 6 samples from sows (between 12 and 36 months old). Additionally, 24 serum samples were collected from ASFV-naïve pigs to serve as negative controls. All sera from ASFV-surviving pigs tested positive while all sera from control pigs tested negative by two different commercial ELISA kits. Antibody reactivity of each serum sample was simultaneously measured against six selected ASFV antigens including p12, p32, p54, pp62, C-type lectin and CD2v. All ASFV-surviving pigs had antibody against p32, p54 and pp62 while 91.3% surviving pigs had antibody against p12. Only small portions of ASFV-surviving pigs exhibited antibodies against C-type lectin (34.8%) and CD2v (26.1%). While antibodies against p12, p32, p54 and pp62 were similarly detected in both finishing pigs and sows, antibodies against C-type lectin and CD2v were mainly detected in sows but not in finishing pigs. These results suggest a differential humoral immune response to ASFV infection in sows and finishing pigs. Further studies are needed to better understand the nature of immune responses to ASFV infection in different pig populations.

The tilapia lake virus (TiLV), a highly infectious negative-sense single-stranded segmented RNA virus, has caused several outbreaks worldwide since its first report from Israel in 2014, and continues to pose a major threat to the global tilapia industry. Despite its economic importance, little is known about the underlying mechanisms in the genomic evolution of this highly infectious viral pathogen. Using phylogenomic approaches to the genome sequences of TiLV isolates from various geographic regions, we report on the pervasive role of reassortment, selection, and mutation in TiLV evolution. Our findings provided the evidence of genome-wide reassortment in this newly discovered RNA virus. The rate of non-synonymous (dN) to synonymous (dS) substitutions was less than one (dN/dS = 0.076 to 0.692), indicating that each genomic segment has been subjected to purifying selection. Concurrently, the rate of nucleotide substitution for each genomic segment was in the order of 1-3 × 10-3 nucleotide substitutions per site per year, which is comparable to the rate of other RNA viruses. Collectively, in line with the results of the previous studies, our results demonstrated that reassortment is the dominant force in the evolution and emergence of this highly infectious segmented RNA virus.

Nowadays, one of the major global health concerns is coronavirus disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Even though numerous treatments and vaccines to combat this virus are currently under development, the detailed molecular mechanisms underlying the pathogenesis of this disease are yet to be elucidated to design future therapeutic tools against SARS-CoV-2 variants. MicroRNAs (miRNAs) are small (20-24 nucleotides), non-coding RNA molecules that regulate post-transcriptional gene expression. Recently, it has been demonstrated that both host and viral-encoded miRNAs are crucial for the successful infection of SARS-CoV-2. For instance, dysregulation of miRNAs that modulate multiple genes expressed in COVID-19 patients with comorbidities (e.g., type 2 diabetes, lung adenocarcinoma, and cerebrovascular disorders) could affect the severity of the disease. Thus, altered expression levels of circulating miRNAs might be helpful to diagnose this illness and forecast whether a COVID-19 patient could develop a severe state of the disease. Besides, researchers have found a number of miRNAs could inhibit the expression of proteins, such as ACE2, TMPRSS2, spike, and Nsp12, involved in the life cycle of SARS-CoV-2. Accordingly, miRNAs represent potential biomarkers and therapeutic targets for this devastating viral disease. Therefore, in this current review, we present the recent discoveries regarding the clinical relevance and biological roles of miRNAs in COVID-19.

Due to the lack of an adaptive immune system, insects rely on innate immune mechanisms to fight against pathogenic infections. Two major innate immune pathways, Toll and IMD, orchestrate anti-pathogen responses by regulating the expression of antimicrobial peptide (AMP) genes. Although the antifungal or antibacterial function of AMPs has been well characterized, the antiviral role of AMPs in insects remains largely unclear. Periplaneta americana (P. americana), or the American cockroach, is used in traditional Chinese medicine as an antiviral agent; however, the underlying mechanism of action of P. americana extracts is unclear. Our previous study showed that the P. americana genome encodes multiple antimicrobial peptide genes. Based on these data, we predicted five novel P. americana defensins (PaDefensins) and analyzed their primary structure, secondary structure, and physicochemical properties. The putative antiviral, antifungal, antibacterial, and anticancer activities suggested that PaDefensin5 is a desirable therapeutic candidate against viral diseases. As the first experimental evidence of the antiviral effects of insect defensins, we also showed the antiviral effect of PaDefensin5 in Drosophila Kc cells and Drosophila embryos in vivo . In conclusion, results of both in silico predictions and subsequent antiviral experiments suggested PaDefensin5 a promising antiviral drug.

Hepatitis E virus (HEV) is a zoonotic agent mainly transmitted through the consumption of uncooked or undercooked meat products derived from infected animals. In Japan, domestic pigs and wild boars are the major animal reservoirs, and whether or not deer are an HEV reservoir remains controversial. We analyzed 395 serum and 199 liver samples from 405 sika deer (Cervus nippon) caught in the wild between 1997 and 2020 in 11 prefectures of Japan for markers of HEV infection. Overall, 17 deer had anti-HEV IgG (4.3%), while 1 (0.2%) had HEV RNA (genotype 3b), indicating the occurrence of ongoing HEV infection in wild deer in Japan. An analysis of the complete HEV genome (deJOI_14) recovered from a viremic deer in Oita Prefecture revealed only 88.8% identity with the first HEV strain in sika deer (JDEER-Hyo03L) in Japan, being closest (96.3%) to the HEV obtained from a hepatitis patient living in the same prefecture. Of note, the deJOI_14 strain was 8.7-9.0% different from the wild boar HEV strains obtained in the same habitat and the same year, suggesting that difference in infected HEV strains between boar and deer may be explained by the limited possibility of close contact with each other, although boars are a known source of HEV infection. Increased numbers of hepatitis E cases after consumption of raw or undercooked meat products of wild deer have been reported in Japan. These results suggest a low but nonnegligible zoonotic risk of HEV infection in wild deer in this country.

The E484K mutation at the SARS-CoV-2 Spike protein emerged independently in different variants around the world and has been widely associated with immune escape from neutralizing antibodies generated during previous infection or vaccination. In this work, the B.1 + L249S+E484K lineage was isolated along with A.1, B.1.420, and B.1.111 SARS-CoV-2 lineages without the E484K mutation and the neutralizing titer of convalescent sera was compared using microneutralization assays. While no significant differences in the neutralizing antibody titers were found between A.1 and B.lineages without the E484K mutation, the neutralizing titers against B.1 + L249S+E484K were 1.5, 1.9, 2.1, and 1.3-fold lower than against A.1, B.1.420, B.1.111-I, and B.1.111-II, respectively. However, molecular epidemiological data indicate that there is no increase in the transmissibility rate associated with this new lineage. This study supports the capability of new variants with the E484K mutation to be resistant to neutralization by humoral immunity, and therefore the need to intensify surveillance programs to determine if these lineages represent a risk for public health.

Alongshan virus (ALSV) is an emerging tick-borne pathogen that infects humans, causing febrile disease. ALSV uses Ixodes Persulcatus ticks to infect humans with a wide range of signs, from asymptomatic to encephalitis-like syndrome. There is an increasing public health concern about the ALSV infection. To get insight into the impacts of viral relations with their hosts on viral ability, survival, and evasion from hosts immune systems remain unknown. The codon usage is a driving force in viral genome evolution; therefore, we enrolled 41 ALSV strains in codon usage analysis to elucidate the molecular evolutionary dynamics of ALSV. The results indicate that the overall codon usage among ALSV isolates is relatively similar and slightly biased. Base compositions for the cds were in order of G >A >C >U and in the third position of codons G3 >A3 >C3 >T3. The RSCU values revealed that the more frequently used codons were mostly GC ended. Different codon preferences in ALSV genes in relation to codon usage of H. sapiens and Ixodes Persulcatus genes were found. Neutrality plot was determined to reveal the superiority of natural selection over directional mutation pressure in causing CUB based on GC12 versus GC3 contents. The results of these studies suggest that the emergence of ALSV in China, Russia and Finland may also be reflected in ALSV codon usage. Altogether, the presence of both mutation pressure and natural selection effect in shaping the codon usage patterns of ALSV.

Synthetic biology has been applied countless times for the modification and improvement of bacterial strains and for the synthesis of products that do not exist in nature. Phages are natural predators of bacteria controlling their population levels; however, their genomes carry several genes with unknown functions. In this work, Bacteriophage Recombineering of Electroporated DNA was used to assess the influence of deletion of a single gene with unknown function in the overall replication parameters of Salmonella phage PVP-SE2. Deletion of ORF_01, transcribed immediately after infection, reduced both the latent and rise periods by 5 min in PVP-SE2ΔORF_01 compared to the wild-type phage. A direct consequence of the deletion led to a smaller progeny release per infected cell by the mutant compared to the wild-type phage. Despite the difference in growth characteristics, the mutant phage remained infective towards exponentially growing cells. The mutation engineered endured for at least ten passages, showing that there is no reversion back to the wild-type sequence. This study provides proof of concept that methodologies used for phage engineering should always be complemented by phage growth characterization to assess whether a mutation can trigger undesirable characteristics.

This study sought to clone and sequence the interferon-γ (IFN-γ) gene of the Fischer's lovebird parrot (Agapornis fischeri). Raw264.7 cells treated with the expressed IFN-γ protein exhibited an upregulation in inducible nitric oxide synthase protein expression and nitric oxide (NO) production coupled with increases in phagocytosis and pinocytosis, as well as an induction of interferon-stimulated genes through the activation of the NF-κB factor, all of which are indicators of the innate immune responses of the activated macrophages. Similar to the IFN-γ protein of other species, the NO production activity of the parrot IFN-γ protein decreased by 80% after exposure at 60 °C for 4 min. Additionally, only half of the NO production activity of the parrot IFN-γ protein remained upon exposure to HCl for 30 min. These findings suggested that the parrot IFN-γ protein was heat-labile and sensitive to acidic conditions. Therefore, all of these effects contributed to the blockage of the uptake of BFDV virus-like particles (VLPs) by cells, the nuclear entry of the Cap protein of BFDV VLPs, and the clearance of the virus from BFDV-infected parrots by the IFN-γ protein of Agapornis fischeri. This study is the first to describe the cloning of the IFN-γ gene of Agapornis fischeri and characterize the anti-beak and feather disease virus activity of the IFN-γ protein of Agapornis fischeri.

A novel Lactobacillus delbrueckii bacteriophage PMBT4 was isolated from the Nigerian fermented milk product nono. The phage possesses a long and thin, non-contractile tail and an isometric head, indicating that it belongs to the Siphoviridae family. A neck passage structure (`collar`), previously hypothesized to be encoded by two genes located in the Lactobacillus delbrueckii phage LL-K insertion sequence (KIS) element, as well as in two additional Lb. delbrueckii phages Ld17 and Ld25A, could also be observed on an estimated 1-5% of phage particles by transmission electron microscopy. However, neither mapping of high throughput sequencing data to KIS element genes from Lb. delbrueckii phages LL-K, Ld17 and Ld25A nor PCR amplification of the KIS element genes could corroborate the presence of these genes in the PMBT4 genome. The PMBT4 genome consists of 31,399 bp with a mol% GC content of 41.6 and exhibits high (95-96%) sequence homologies to Lb. delbrueckii phages c5, Ld3, Ld25A and Ld17, which assigned PMBT4 as a new member of this genus, i.e. the Cequinquevirus genus.

In 2020, to trace the prevalence and evolution of bovine coronavirus (BCoV) in China, a total of 1383 samples (1016 fecal samples and 367 nasal swab samples) were collected from 1016 cattle exhibiting diarrhea symptoms on dairy farms and beef cattle farms in Heilongjiang Province, Northeast China. All samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) detection of the BCoV N gene, followed by an analysis of its epidemiology and genetic evolution. The results indicated that of the 1016 diarrhea-affected cattle, 15.45% (157/1016) were positive for BCoV, in which positive rates of the fecal and nasal swab samples were 12.20% (124/1016) and 21.53% (79/367), respectively. Of the 367 cattle whose nasal swab samples were collected, the BCoV positive rate of the corresponding fecal samples was 15.26% (56/367). BCoV infection was significantly associated with age, farming pattern, cattle type, farm latitude, sample type, and clinical symptom (p < 0.05). Of the 203 BCoV-positive samples, 20 spike (S) genes were successfully sequenced. The 20 identified BCoV strains shared nucleotide homologies of 97.7-100.0%, and their N-terminal domain of S1 subunit (S1-NTD: residues 15-298) differed genetically from the reference strains of South Korea and Europe. The 20 identified BCoV strains were clustered in the Asia-North America group (GII group) in the global strain-based phylogenetic tree and formed three clades in the Chinese strain-based phylogenetic tree. The HLJ/HH-10/2020 strain was clustered into the Europe group (GI group) in the S1-NTD-based phylogenetic tree, exhibiting N146/I, D148/G, and L154/F mutations that affect the S protein structure. Of the identified BCoV strains, one potential recombination event occurred between the HLJ/HH-20/2020 and HLJ/HH-10/2020 strains, which led to the generation of the recombinant BCV-AKS-01 strain. A selective pressure analysis on the S protein revealed one positively selected site (Asn509) among the 20 identified BCoV strains located inside the putative receptor binding domain (residues 326-540). These data provide a greater understanding of the epidemiology and evolution of BCoV in China.

Picobirnaviruses (PBVs) are bi-segmented dsRNA viruses that have been detected in various animal species including vertebrates and invertebrates. In this study, 17 complete or incomplete PBV segment-2 and one unsegmented PBV-like virus sequence were identified in fecal samples from different bird species using viral metagenomic approach. The bird PBV and PBV-like virus retained the conservative motifs that are conserved in dsRNA2 of common PBVs. The RdRp of these 17 PBVs shared the highest Amino acid (aa) identity of 45.90%∼94.19% with previous animal and human PBVs, while the RdRp of the unsegment PBV-like virus shared the highest aa sequence identity of 31.93% with one chicken PBV (GenBank No. MW837829). The unsegmented PBV-like virus unexpectedly used the yeast mitochondrial genetic code (transl_table=3) for all ORFs translation. In addition, the prokaryotic RBS sequence was not only detected upstream to ORF2 at position 360AGGAGG365 of this unsegmented PBV-like virus, but also found upstream to ORF of bird PBV dsRNA2. The presence of the prokaryotic ribosomal binding site in the bird PBV genomes, and the finding of one novel unsegmented PBV-like virus using the yeast mitochondrial genetic code for translation supported recent speculations that PBVs may actually infect prokaryotic or fungal host cells. This study enhanced our understanding of PBVs and provided data support for exploring the real host of PBVs.

Avian polyomavirus (APV) is a non-enveloped virus with a circular double-stranded DNA genome approximately 5000 bp in length. APV was first reported in fledgling budgerigars (Melopsittacus undulatus) as the causative agent of budgerigar fledgling disease, resulting in high parrot mortality rates in the 1980s. This disease has been observed worldwide, and APV has a wide host range including budgerigars, cockatoos, lorikeets, lovebirds, and macaws. Twenty APV isolates have been collected from healthy and symptomatic parrots in Taiwan from 2015 to 2019. These isolates were then amplified via polymerase chain reaction, after which the whole genomes of these isolates were sequenced. The overall APV-positive rate was 14.2%, and the full lengths of the APV Taiwan isolates varied from 4971 to 4982 bps. The APV genome contains an early region that encodes two regulatory proteins (the large tumor antigen (Large T-Ag) and the small tumor antigen (Small t-Ag)) and a late region which encodes the capsid proteins VP1, VP2, VP3, and VP4. The nucleotide identities of the VP1 and VP4 genes ranged from 98.7 to 100%, whereas the nucleotide sequence of the Large T-Ag gene had the highest identity (99.2-100%) relative to other APV isolates from the GenBank database. A phylogenetic tree based on the whole genome demonstrated that the APV Taiwan isolates were closely related to Japanese and Portuguese isolates. Recombination events were analyzed using the Recombination Detection Program version 4 and APV Taiwan isolate TW-3 was identified as a minor parent of the APV recombinants. In this study, we first reported the characterization of the whole genome sequences of APV Taiwan isolates and their phylogenetic relationships with all APV isolates available in the GenBank database.

Dendrobium officinale is an important traditional Chinese medicinal herb, and the stem tissue is the main medicinal that is harvested from D. officinale. Recently, the first viroid was identified from D. officinale in China, and it has been named Dendrobium viroid (DVd). Whether DVd interferes with metabolic pathways in dendrobium plants and affects the medicinal value of the host is unknown. In this study, metabolomics data from stem tissues supported by transcriptome studies were used to investigate how metabolism modulate of D. officinale is altered by DVd infection. Our results show that metabolism of D. officinale is reprogrammed in many ways during DVd infection, and this is reflected by significant changes in the levels of flavonoids, alkaloids, and phenolic acids. Furthermore, we found that DVd infection significantly decreased the accumulation of flavonoids and alkaloid metabolites in infected stems, and the decreases in these metabolites appears to affect the medicinal components of the infected plants, weakening the host antiviral immune response as well. Conversely, phenolic acids occupy a larger proportion of the up-regulated metabolites from DVd infection in comparison with the mock-inoculated control, and the increase in the total phenolic acids may reflect the activation of the pathogen defense response in D. officinale. Taken together, our results provide an interesting overview and give a better understanding of the relationship between metabolism and DVd infection in the orchid D. officinale.

BACKGROUND: COVID-19 has posed unforeseen circumstances and throttled major economies worldwide. India has witnessed two waves affecting around 31 million people representing 16% of the cases globally. To date, the epidemic waves have not been comprehensively investigated to understand pandemic progress in India. OBJECTIVE: Here, we aim for pan Indian cross-sectional evolutionary analysis since inception of SARS-CoV-2. METHODS: High quality genomes, along with their collection date till 26th July 2021, were downloaded. Whole genome-based phylogeny was obtained. Further, the mutational analysis was performed using SARS-CoV-2 first reported from Wuhan (NC_045512.2) as reference. RESULTS: Based on reported cases and mutation rates, we could divide the Indian epidemic into seven phases. The average mutation rate for the pre-first wave was <11, which elevated to 17 in the first wave and doubled in the second wave (∼34). In accordance with mutation rate, VOCs and VOIs started appearing in the first wave (1.5%), which dominated the second (∼96%) and post-second wave (100%). Nation-wide mutational analysis depicted >0.5 million mutation events with four major mutations in >19,300 genomes, including two mutations in coding (spike (D614G), and NSP 12b (P314L) of rdrp), one silent mutation (NSP3 F106F) and one extragenic mutation (5' UTR 241). CONCLUSION: Whole genome-based phylogeny could demarcate post-first wave isolates from previous ones by point of diversification leading to incidences of VOCs and VOIs in India. Such analysis is crucial in the timely management of pandemic.

The oak lappet moth, Trabala vishnou gigantina is a forest insect pest that damages broad-leaf trees severely. Trabala vishnou gigantina nucleopolyhedrovirus (TrviNPV) has been isolated from a naturally infected T. vishnou gigantina larva and investigated for its biology and the potential to be a biological control agent against its insect host. TrviNPV was characterized by electron microscope of occlusion bodies (OBs), genomic sequencing and field control efficacy. TrviNPV OBs exhibited an irregular polyhedral shape varying in size from 0.99 to 3.99 μm with multiple nucleocapsids per virion. The genome of this virus was 165 657 bp in length with 40.33% GC content and encoded 146 putative ORFs including the 38 baculovirus core genes. TrviNPV is a group II alphabaculovirus that encodes F protein and lacks the gp64 gene specific to group I alphabaculoviruses. Phylogeny and Kimura-2 parameter analysis revealed TrviNPV to be a novel species and closest to ArdiNPV, EupsNPV and OrleNPV. Bioassays and field trials in a shrubland revealed that TrviNPV was virulent and effective to control T. vishnou gigantina in arid semi-desert region. This work firstly reported the whole genome of TrviNPV as well as its biological characters for a possibility to develop this virus as bio-pesticide in the future.

The complete genome (2298 nucleotides) of the economically important and immunosuppressive, chicken infectious anemia virus (CAV), from a disease outbreak in a layer flock is discussed. This is the first report of a complete genome sequence of CAV from India. The phylogenetic analyses grouped this isolate with CAV genogroup IIIb based on both complete genome and capsid protein (VP1) sequences. The analyses further revealed the presence of CAV genogroups II, IIIa and IIIb in India. The VP1 sequence identity ranged between 84.4 to 99.3% with that of the Indian isolates and carried a unique substitution at position 447 (serine instead of threonine). Two novel amino acid substitutions were observed at position 52 of VP1 (serine instead of proline) and at position 26 of VP2 (asparagine instead of serine). Sequence analyses of VP1, VP2 and VP3 suggested that the isolate could be attenuated. Comparison with CAV variants, isolated from mammalian species, showed similarities in the numbers of certain transcription factor binding sites in the non-coding regions. Recombination analysis detected no recombination events in this isolate. Further investigations are needed to understand the implications of the unique features of this isolate on viral virulence.

The SARS-CoV-2 alpha VOC (also known as lineage B.1.1.7) initially described in the autumn, 2020 in UK, rapidly became the dominant lineage across much of Europe. Despite multiple studies reporting molecular evidence suggestive of its circulation in Brazil, much is still unknown about its genomic diversity in the state of São Paulo, the main Brazilian economic and transportation hub. To get more insight regarding its transmission dynamics into the State we performed phylogenetic analysis on all alpha VOC strains obtained between February and August 2021 from the Sao Paulo state Network for Pandemic Alert of Emerging SARS-CoV-2 variants. The performed phylogenetic analysis showed that most of the alpha VOC genomes were interspersed with viral strains sampled from different Brazilian states and other countries suggesting that multiple independent Alpha VOC introductions from Brazil and overseas have occurred in the São Paulo State over time. Nevertheless, large monophyletic clusters were also observed especially from the Central-West part of the São Paulo State (the city of Bauru) and the metropolitan region of the São Paulo city. Our results highlight the Alpha VOC molecular epidemiology in the São Paulo state and reinforce the need for continued genomic surveillance strategies for the real-time monitoring of potential emerging SARS-CoV-2 variants during the ever-growing vaccination process.

The increase of multi-drug resistant and multi-serotypes of pathogenic Escherichia coli has brought more severe challenge to control infection. Nowadays, bacteriophage is a promising tool to treat colibacillosis as an alternative of antibiotics. A coliphage P479, isolated from sewage of poultry farm, could lyse multiple serotypes, including not only O1, O2, O8, O9, O21, O78, O83, O145 of Avian pathogenic E. coli, but O157:H7 of Enterohaemorrhagic E. coli and O18:K1:H7 Neonatal meningitis E. coli. Additionally, P479 could also lyse multi-drug resistant E. coli. These indicated that P479 had good lytic ability. One-step growth curve revealed that the latent time period of P479 was 10 min and the burst size was about 318 PFU/cell. Stability tests demonstrated that P479 had good stability under various temperature (4 to 50 °C) and pH (3 to11) conditions. P479 contained of a linear, double-stranded DNA molecule of 172,033 bp with 40.3% GC content. P479 contained 296 putative coding sequences (CDSs) and two tRNA genes. Based on genomic comparison, P479 was classified as a member of genus Gaprivervirus, subfamily Tevenvirinae, family Myoviridae, order Caudovirales. No known virulent or lysogenic genes were detected in the genome of P479, manifesting P479 was safe to adhibit. Antibacterial activity in vitro manifested that P479 has varying degrees bacteriostatic activity against different bacteria. According to the above properties, P479 has the potential to be applied in phage therapy in the future.

Since June 2015, Fowl adenovirus outbreaks have occurred in China, causing significant economic losses to poultry industry. The FAdV-4 Fiber-2 proteins could induce effective protection, but the precise mechanism of immune protection remains unknown. Here, we have compared the biological characteristics of Fiber-2 protein of the very virulent WZ strain of FAdV-4 (vvFAdV-4) with that of non-virulent ON1 strain. The sequence analysis revealed natural deletions and sequence differences between the classical non-pathogenic strain ON1 and the vvFAdV-4 isolate. These two Fiber-2 proteins successfully expressed in E. coli resemble in structure and function to the native-like trimeric protein. The trimeric structure and bioreactivity of the recombinant Fiber-2 proteins to FAdV-4 specific antibodies were characterized. The immune protection induced by Fiber-2 proteins of FAdV-4 WZ and ON1 strains were compared in SPF chickens. All birds in the WZ-Fiber-2 immunized group generated systemic specific antibodies compared with both ON1-Fiber-2 protein and PBS immunized groups. According to the results of attack mortalities, viral shedding and tissue gross lesion, the WZ Fiber-2 protein induced complete protection at a dose of 2 μg per chicken, whereas the ON1-Fiber-2 protein induced 0 protection at 3 dpc. In view of the characteristics of Fiber-2 proteins of different strains, this study can help us to further understand the mechanism of protective immunity and provide a basis for the prevention and control of FAdV-4 in chickens.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) undergoes a biphasic life cycle with the production of two physically and functionally distinct virions: budded virions (BVs) and occlusion-derived virions (ODVs). Nuclear egress of nucleocapsids and intranuclear microvesicle formation are critical for the morphogenesis of BVs and ODVs, respectively, but the mechanisms and details of these two processes remain unknown. Our previous studies have shown that AcMNPV p48 (ac103) gene is essential for the nuclear egress of nucleocapsids and efficient formation of intranuclear microvesicles, and protein P48 associates with Ac93, which is also involved in the above processes in virion morphogenesis. In this study, we present evidence that alanine substitution for residues N318, V319, C320, R321, and I323 of P48 disrupted the association with Ac93. Moreover, mutation of these residues blocked the nuclear egress of nucleocapsids and efficient formation of intranuclear microvesicles, and subsequent BV formation, as well as ODV envelopment and embedding of ODVs into polyhedra. These results suggested that the association between P48 and Ac93 may be important for both BV and ODV morphogenesis.

TRIM5γ, together with TRIM31, has been shown to promote HBx ubiquitination and degradation. This study aimed to explore whether a patient with HCC (hepatic cell carcinoma) having a small nucleotide inserted into the TRIM31 gene, which made a shorter transcript stop at 768 bp, would result in blocking the activity of TRIM31 in promoting HBx degradation. Besides, this study aimed to determine the binding region of the TRIM31-TRIM5γ-HBx complex. HBV (Hepatitis B virus) infection was reported to induce type-III IFN but not type-I or type-II IFNs, here TRIM31 was found to be a type III rather than a type I stimulated gene, which was indispensable in inhibiting the hepatitis B virus replication by the interferon families. Thus, this study further identified the critical role of TRIM31 in the host-hepatitis B virus interaction.

An 8-month-old child diagnosed with severe combined immunodeficiency (SCID) was found to be excreting vaccine-derived poliovirus (VDPVs). Five stool samples from the child and stool samples from 24 contacts were collected during the following 7 months. Complete genome sequence by next generation sequencing (NGS) identified 0.7 to 1.4% nucleotide substitutions in the capsid P1 region of the first and the last isolates compared with Sabin 3 strain. Simplot analysis revealed that all isolates were Sabin 3/Sabin 1 recombinants, sharing a single recombination breakpoint in the 2C region. Multiple nucleotide variants were identified in the 5'UTR (T472→C and G395→A); amino acid mutations were identified in residues at VP1-6 (Thr to Ile), VP1-105 (Met to Thr), VP1-286 (Arg to Lys), VP2-155 (Lys to Glu), VP3-59 (Ser to Asn) and VP3-91 (Phe to Ser). These variants were commonly observed in other PV strains, which may contribute to attenuation and temperature sensitivity. None of the 24 tested contacts of the patient and related transmits was found to be infected with poliovirus. Our study provides a rapid and reliable method for the characterization of VDPV research in Poliovirus infection. In post-OPV era, immunodeficient people with persistent and chronic infection remain a major challenge for polio eradication in China.

Bats are important reservoirs for many kinds of emerging zoonotic viruses. In order to explore potential pathogens carried by bats and trace the source of adenovirus outbreaks on the southeastern coast of China, we took pharyngeal and anal swabs from a total of 552 bats (Rhinolophus pusillus) collected from various areas of Chinese southeastern coast. Adenoviruses were identified in 36 out of the 552 samples (6.5%) . Complete genome sequences of two adenovirus isolations from Vero E6 cells were obtained, which were further validated as identical strains via next-generation sequencing and were named Bat-Advcxc6. The cell culture inoculated with the two samples exhibited remarkable cytopathic changes. The full genome has 37,315 bp and owns 29 open reading frames. Phylogenetic analyses confirmed that Bat-Advcxc6 represented a novel bat adenovirus species in the genus Mastadenovirus. Transmission electron microgram showed clear virus particles. Bat-Advcxc6 shared similar characteristics of G + C contents with Bat mastadenovirus WIV11 (Bat mastadenovirus C) found in China in 2016, but differed from this serotype due to a <75% similarity with DNA polymerase amino acid sequences in WIV11. As it is a newly found adenovirus strain according to the international classification criteria, further analyses of virus dynamics, epithelial invasion, and immunization assays are required to explore its potential threats of cross-species transmission.